The objective of this research is a detailed investigation of the structure and function of viral RNA folding motifs involved in ribosomal frameshifting. In many viruses, including tumor- and retro- viruses, the programmed-1 ribosomal frameshifting of polycistronic mRNA regulates the relative level of structural and enzymatic proteins important for efficient viral assembly. The - 1 shift in reading frames causes stop codon readthrough, and results in production of a single fusion protein. For example, in the Rous sarcoma retrovirus, the pol gene that encodes integrase, protease and reverse transcriptase is expressed with the upstream gag gene (encoding virus core proteins) through a gag-pol fusion protein. The mature products are later obtained by processing the poly-protein precursor. The -1 frameshifting is not only found in retroviruses, but also found in coronaviruses, yeast and plant viruses, as well as bacterial systems. Frameshifting levels can range from 1 to over 30 percent in different systems to produce gene products in a functionally appropriate ratio. It is postulated that a complex mRNA structure 6-8 nucleotides downstream from the "slippery sequence", in many cases a pseudoknot, leads to ribosomal pausing and the simultaneous slippage of both aminoacyl and peptidyl tRNAS toward the 5' direction by one base. The slippery shift site on the messenger RNA has an X XXY YYN consensus sequence (the initial reading frame is indicated, and bases X and Y can be identical). However, the mechanism of ribosomal frameshifting is not understood. For example, it remains to be determined how individual conformational features of the pseudoknot motif affect the relative level of frameshifting activity. The combined structure and function analysis proposed in this grant application will provide insights into the basis of viral ribosomal frameshifting. The specific aims of this research are: 1) Refinement of the trigonal crystal form of the RNA pseudoknot from beet western yellow virus (BWYV) at 1.3 Angstrom units resolution and its comparison with the structure of the same RNA in a cubic crystal form determined to 2.8 Angstrom units resolution. 2) Crystallization and structure determination of several mutant BWYV pseudoknots that produce either significantly lower or higher frameshifting activity relative to the wt- pseudoknot. 3) Crystallization and structure determination of RNA pseudoknots from potato leaf roll virus (PLRV), mouse mammary tumor virus (MMTV) and simian retrovirus (SRV), a frameshifting stem-loop motif from HIV-I retrovirus (HIVRV) and a hybrid of the SRV and BWYV pseudoknots. 4) Analysis of the frameshifting activities in rabbit reticulocyte lysates and in vivo produced by BWYV RNA pseudoknots with mutations in key structural elements, including the loops crossing the major and minor grooves, and the junction regions between the two stems. 5) Similar mutational analyses of the PLR, MMT and SR viruses to probe the influence of individual elements on the frameshifing activity. 6) Crosslinking studies that will allow the isolation, purification and characterization of proteins that bind to the viral RNA pseudoknots.